Cell and gene cancer therapies require ex vivo cell processing of human grafts. Such processing requires at least three steps - cell enrichment, cell separation (destruction), and gene transfer - each of which requires the use of a separate technology. While these technologies may be satisfactory for research use, they are of limited usefulness in the clinical treatment setting because they have a low processing rate, as well as a low transfection and separation efficacy and specificity in heterogeneous human grafts. Most problematic, because current technologies are administered in multiple steps - rather than in a single, multifunctional, and simultaneous procedure - they lengthen treatment process and introduce an unnecessary level of complexity, labor, and resources into clinical treatment; all these limitations result in high losses of valuable cells. The project goal is to develop a universal, high-throughput, and multifunctional technology that will simultaneously (1) transfect target T cells, (2) destroy unwanted T regulatory cells, and (3) preserve valuable non-target cells in heterogeneous grafts. Each of these functions will have single target cell specificity, hih efficacy, and low toxicity, and will be achieved in a single, high-throughput procedure to meet the clinical requirements for processing human graft ex vivo. Proposed technology employs novel cellular agents, called plasmonic nanobubbles (PNBs). PNBs are not particles, but transient, intracellular events, a vapor nanobubbles that expand and collapse in mere nanoseconds. PNB mechanism supports the hypothesis that treating human grafts with PNBs generated by two different types of gold nanoparticles (and consequently two different PNBs) with a low-energy laser pulse will simultaneously accomplish three following functions: (1) Transfect CD3+ T cells by injecting free external plasmid with small PNBs, (2) Destroy CD25+ T-regulatory cells mechanically with large PNBs and (3) Preserve CD3- and CD25- accessory (non-target) cells. Our extensive preliminary data indicate that PNB technology offers this unprecedented, simultaneous three-in-one functionality, and our team will test this hypothesis through two specific aims: Aim 1. Optimize targeting of human peripheral blood mononuclear cells (PBMC) with gold nanoparticle conjugates to achieve simultaneous generation of small injective PNBs in CD3+ T-cells and large destructive PNBs in CD25+ T-regulatory cells in response to a laser pulse. Aim 2. Adopt the optimized PNB method to enable simultaneous gene transfer in CD3+ T cells and destruction of CD25+ T-regulatory cells without damaging non-target cells in one bulk flow procedure with high throughput. The multi-functionality, precision, and high throughput of all-in-one PNB technology will tremendously impact cell and gene therapies and other clinical applications that depend on ex vivo processing of heterogeneous cell systems.